Determination of immuno-reactive rabbit apolipoprotein B-48 in serum by ELISA.

Apolipoprotein B-48 (apoB-48) is produced by the small intestine, is associated with chylomicrons, and appears to be a suitable marker for clinical studies of postprandial dynamics of lipoproteins. It is also associated with cardiovascular risk factors. We have developed an assay system to quantify immuno-reactive apoB-48 in rabbit serum or plasma. A microtiter-plate was coated with monoclonal antibody raised against human apoB-48 C-terminal specific decapeptide that has high homology to the rabbit C-terminal sequence. Appropriate ELISA standard curves were obtained using apoB-48 extracted from rabbit serum by immuno-affinity chromatography. No cross-reactivity was found with apoB-100 in western blot analyses. Intra- and inter-assay CVs were less than 3%. Recovery of rabbit apoB-48 spiked in serum was within 93.4-105%. ApoB-48 levels in healthy controls rabbits fed a normal diet were within the range of 0.903-1.09 microg/ml (mean +/- SD: 1.03 +/- 0.084 microg/ml). In healthy animals, the blood apoB-48 level was markedly increased by a high fat diet and in the postprandial state in parallel with serum triglyceride. Ezetimibe, cholesterol absorption inhibitor, given orally to rabbits fed on a high fat diet blocked further increase of blood levels of apoB-48 and triglyceride. This method for measuring apoB-48 using the monoclonal antibody is simple, reliable, and suitable for routine analyses.

Cinnamon extract attenuates TNF-alpha-induced intestinal lipoprotein ApoB48 overproduction by regulating inflammatory, insulin, and lipoprotein pathways in enterocytes.

We have previously reported that the obesity-associated proinflammatory cytokine, TNF-alpha, stimulates the overproduction of intestinal apolipoprotein (apo) B48 containing lipoproteins. In the current study, we have evaluated whether a water-soluble cinnamon extract [CE (Cinnulin PF)] attenuates the dyslipidemia induced by TNF-alpha in Triton WR-1339 treated hamsters, and whether CE inhibits the oversecrection of apoB48-induced by TNF-alpha in enterocytes in a 35S labeling study. In vivo, oral treatment of Cinnulin PF (50 mg per kg BW), inhibited the postprandial overproduction of apoB48-containing lipoproteins and serum triglyceride levels. In ex vivo 35S labeling studies, CE (10 and 20 microg/ml) inhibited the oversecretion of apoB48 induced by TNF-alpha treated enterocytes into the media. To determine the molecular mechanisms, TNF-alpha treated primary enterocytes isolated from chow-fed hamsters, were incubated with CE (10 microg/ml), and the expression of the inflammatory factor genes, IL1-beta, IL-6, and TNF-alpha, insulin signaling pathway genes, insulin receptor (IR), IRS1, IRS2, phosphatidylinositol 3-kinase (PI3-K), Akt1 and phosphatase and tensin homology (PTEN), as well as the key regulators of lipid metabolism, cluster of differentiation (CD)36, microsomal triglyceride transfer protein (MTTP), and sterol regulatory element binding protein (SREBP)-1c were evaluated. Quantitative real-time PCR assays showed that CE treatment decreased the mRNA expression of IL-1beta, IL-6 and TNF-alpha, improved the mRNA expression of IR, IRS1, IRS2, PI3K and Akt1, inhibited CD36, MTTP, and PTEN, and enhanced the impaired SREBP-1c expression in TNF-alpha treated enterocytes. These data suggest that a water extract of cinnamon reverses TNF-alpha-induced overproduction of intestinal apoB48 by regulating gene expression involving inflammatory, insulin, and lipoprotein signaling pathways. In conclusion, Cinulin PF improves inflammation related intestinal dyslipidemia.